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Market Survey 2020: Commercial Clinical Decision Support Systems and Wellness Tools

For long-duration, deep space exploration missions, current methods for managing and supporting crew health and medical conditions will be unsuitable. Communication and data transmission lags will necessitate the use of a sophisticated clinical decision support system (CDSS) that will tailor diagnosis and treatment guidance that is context-sensitive for anticipated astronaut health, wellness, and medical conditions. A variety of clinical decision support (CDS) and wellness tools (WT) are currently available in the commercial market and a broad-brush survey of this market can provide an initial impression of the current state of the art which, in turn, can inform the roadmap of NASA deep space CDSS development and associated requirements. Such a survey was undertaken during the first six months of 2020 using directed convenience sampling to obtain information provided by vendors on their websites; both commercially available CDS and WT (such as those used to track and monitor nutrition, exercise, and sleep) were included. Areas assessed were item type (e.g., software/application, device); primary purpose of the item (e.g., diagnostic support, nutrition tracking); additional purposes (if any); reported features, capabilities, and functionality; setting of use (e.g., inpatient, outpatient); intended user (e.g., clinician, patient); location and sources of data/information used or produced by the item; integration with patient electronic health record (EHR); compliance with interoperability ontologies and standards (e.g., Health Level 7 [HL7], Systematized Nomenclature of Medicine – Clinical Terminology [SNOMED-CT]); and whether the item is knowledge-based (derived from research findings) or non-knowledge-based (derived through artificial intelligence, machine learning, advanced probability and statistics), among others. Ninety-seven (97) vendor websites describing 196 CDS and 73 WT (269 total) were reviewed and coded. The primary purpose of the majority of CDS reviewed is diagnosis or diagnosis/treatment/drug decision support—targeted for clinician use— and the primary purpose of the majority of WT reviewed is the monitoring of different health metrics, most often through the use of a biosensor device (e.g., blood pressure)—targeted for patient use. Very few CDS or WT appear to comply with major international interoperability standards or can be integrated with a patient’s EHR data. None consider contextual factors, such as conditions of the physical environment (e.g., CO2 levels). The majority of CDS and WT reviewed are non-knowledge, cloud- or web-based applications or software. Forty-three (43) major findings were identified and the implications those findings have for NASA will be discussed. Example major findings include: CDS-WT capabilities range from diagnosis to treatment applications, CDS-WT may be wearable or non-wearable and are technologically advanced and only a few CDS-WT tools referenced compliance to ensure interoperability, among other findings. Recommendations will also be offered that will help to address ExMC Gap, Medical-701: Enhance medical capabilities within an exploration medical system.

market survey↗

Establishing Regulatory Jurisdictional Boundaries at Collocated Advanced-Reactor Facilities

This white paper discusses establishing and applying nuclear facility jurisdictional boundaries at advanced-nuclear-reactor facilities. It was written for industry review and evaluation with possible consideration by the U.S. Nuclear Regulatory Commission (NRC) for subsequent regulatory action. The paper proposes a regulatory basis for establishing jurisdictional boundaries at operational advanced (i.e., non-light water) reactor installations at sites proximate to and sharing systems with non-NRC regulated facilities (e.g., users of process heat, fossil-plant retrofits, microgrid electrical power, desalination, etc.). Advanced-reactor technologies can be applied to support many industrial applications to replace the burning of fossil fuels as well as produce steam for electricity. The principal application of the current light-water reactor fleet is electricity generation. These varied industrial applications may involve an advanced-reactor design in combination with different site-specific energy-conversion systems. Some of these process-heat applications will require process-heat delivery systems to lie partially outside the advanced-reactor operator’s facility. Energy conversion systems are conventional, non-nuclear equipment and buildings. Given these varied applications, there should be a clear understanding between the advanced-reactor applicant and the NRC regarding a nominal demarcation between those systems that reside within the nuclear facility under the regulatory jurisdiction of the NRC (i.e., within the scope of a 10 Code of Federal Regulations (CFR) 50 operating license or Part 52 design certification and a combined license) and those that fall outside the scope of the NRC (e.g., an industrial facility). Additionally, it is important to have a clear understanding regarding the plant scope that should be addressed in an advanced-reactor facility Part 52, design certification application and the part of the plant scope that could be addressed as part of a site specific combined license application.

11 NUCLEAR FUEL CYCLE AND FUEL MATERIALS↗

optimas v0.1

optimas is a Python library that can coordinate a large number of numerical simulations on high-performance computing resources, with the aim of optimizing a given simulation result. More specifically, this library is currently used for design optimization of laser-plasma particle accelerators. In this context, the performance of particle accelerator designs are often evaluated with large-scale simulation codes, and many separate simulations need to be run - with different design parameters - in order to find the most performant design. optimas facilitates this process by providing a convenient interface to advanced optimizers (e.g. Bayesian optimization), and by coordinating the execution of the different simulations on HPC resources. (This is done by leveraging the library libensemble.) Compared to other open-source optimization libraries (e.g. Ax), optimas is more tailored towards execution on DOE HPC resources (e.g. Perlmutter, Summit, etc.) and is specialized for the type of simulation codes and workflows that are used in the community of laser-plasma acceleration.

Lehe, Remi↗

Overview: Small Aircraft Transportation System Airborne Remote Sensing Fuel Droplet Evaporation

The NASA Nebraska Preparation Grant was designed to solidify relationships, intensify communication, and launch collaborative initiatives among Nebraska researchers and key contacts at NASA research centers and enterprises. In doing so, Nebraska was successful in laying the groundwork for the foundation for numerous long-term, mutually beneficial collaborations that were subsequently proposed and awarded in the NASA EPSCoR 2000 competition. The NASA Nebraska EPSCoR Preparation Grant was managed by the same administrative team that oversees Nebraska's NASA Space Grant and EPSCoR programs. An advisory board (later Technical Advisory Committee) made up of voting representatives from all affiliate and partner organizations regularly reviewed grant progress and direction. The University of Nebraska at Omaha's Aviation Institute, the host institution for all three programs, provided additional administrative oversight and program evaluation through established review mechanisms. This structure has served NASA well and has been cited as a model program. The second year of preparation grant funding served as a significant opportunity for Nebraska to lay the groundwork for the continued elevation and success of its NASA EPSCoR program. In anticipation of the NASA EPSCoR 2000 grant competition, Year 2 funding enabled funded researchers to further broaden and enhance the quality and quantity of collaborations with NASA Field Centers, Codes, and Enterprises. The plan set the stage for long-term research and outreach endeavors that have contributed significantly to the achievement of NASA's strategic objectives; the state of Nebraska's economic and aerospace development efforts; and have advanced Nebraska s aeronautics research efforts to a national leadership level. The overarching goal of the NASA Nebraska EPSCoR Preparation grant was met by facilitating research endeavors among Nebraska faculty that addressed research and technology priorities of the NASA Field Centers, Codes, and Strategic Enterprises. During the first year of funding, Nebraska established open and frequent lines of communication with university affairs officers and other key personnel at all NASA Centers and Enterprises, and facilitated the development of collaborations between and among junior faculty in the state and NASA researchers. As a result, Nebraska initiated a major research cluster, the Small Aircraft Transportation System Nebraska Implementation Template.

Bowen, Brent↗

Unstructured Mesh Methods for the Simulation of Hypersonic Flows

This report describes the research work undertaken at the Massachusetts Institute of Technology. The aim of this research is to identify effective algorithms and methodologies for the efficient and routine solution of hypersonic viscous flows about re-entry vehicles. For over ten years we have received support from NASA to develop unstructured mesh methods for Computational Fluid Dynamics. As a result of this effort a methodology based on the use, of unstructured adapted meshes of tetrahedra and finite volume flow solvers has been developed. A number of gridding algorithms flow solvers, and adaptive strategies have been proposed. The most successful algorithms developed from the basis of the unstructured mesh system FELISA. The FELISA system has been extensively for the analysis of transonic and hypersonic flows about complete vehicle configurations. The system is highly automatic and allows for the routine aerodynamic analysis of complex configurations starting from CAD data. The code has been parallelized and utilizes efficient solution algorithms. For hypersonic flows, a version of the, code which incorporates real gas effects, has been produced. One of the latest developments before the start of this grant was to extend the system to include viscous effects. This required the development of viscous generators, capable of generating the anisotropic grids required to represent boundary layers, and viscous flow solvers. In figures I and 2, we show some sample hypersonic viscous computations using the developed viscous generators and solvers. Although these initial results were encouraging, it became apparent that in order to develop a fully functional capability for viscous flows, several advances in gridding, solution accuracy, robustness and efficiency were required. As part of this research we have developed: 1) automatic meshing techniques and the corresponding computer codes have been delivered to NASA and implemented into the GridEx system, 2) a finite element algorithm for the solution of the viscous compressible flow equations which can solve flows all the way down to the incompressible limit and that can use higher order (quadratic) approximations leading to highly accurate answers, and 3) and iterative algebraic multigrid solution techniques.

Peraire, Jaime↗

Advanced Methods for Aircraft Engine Thrust and Noise Benefits: Nozzle-Inlet Flow Analysis

The Fluid Mechanics and Acoustics Laboratory at Hampton University (HU/FM&AL) jointly with the NASA Glenn Research Center has conducted four connected subprojects under the reporting project. Basically, the HU/FM&AL Team has been involved in joint research with the purpose of theoretical explanation of experimental facts and creation of accurate numerical simulation techniques and prediction theory for solution of current problems in propulsion systems of interest to the NAVY and NASA agencies. This work is also supported by joint research between the NASA GRC and the Institute of Mechanics at Moscow State University (IM/MSU) in Russia under a CRDF grant. The research is focused on a wide regime of problems in the propulsion field as well as in experimental testing and theoretical and numerical simulation analyses for advanced aircraft and rocket engines. The FM&AL Team uses analytical methods, numerical simulations and possible experimental tests at the Hampton University campus. The fundamental idea uniting these subprojects is to use nontraditional 3D corrugated and composite nozzle and inlet designs and additional methods for exhaust jet noise reduction without essential thrust loss and even with thrust augmentation. These subprojects are: (1) Aeroperformance and acoustics of Bluebell-shaped and Telescope-shaped designs; (2) An analysis of sharp-edged nozzle exit designs for effective fuel injection into the flow stream in air-breathing engines: triangular-round, diamond-round and other nozzles; (3) Measurement technique improvement for the HU Low Speed Wind Tunnel; a new course in the field of aerodynamics, teaching and training of HU students; experimental tests of Mobius-shaped screws: research and training; (4) Supersonic inlet shape optimization. The main outcomes during this reporting period are: (l) Publications: The AIAA Paper #00-3170 was presented at the 36th AIAA/ASME/SAE/ASEE Joint Propulsion Conference, 17-19 June, 2000, Huntsville, AL. The AIAA Paper #01-1893 has been accepted for the AIAA/NAL-NASDA-ISAS 10th International Space Planes and Hypersonic Systems and Technologies Conference, 24-27 April 2001, Kyoto, Japan. The AIAA Paper #01 -3204 has been accepted for presentation at the 37th AIAA/ASME/SAE/ASEE Joint Propulsion Conference, being held on 08-11 July, in Salt Lake City, UT; (2) A U.S. patent #6,082,635 was granted on July 4, 2000; (3) Grants and proposals: The H U/ FM&AL was awarded the NASA grant NAG-3-2495 in October 2000 and the laboratory is a primary U.S. research team in a joint project under the CRDF award granted to the NASA GRC and IM/MSU (Russia) in July 2000; (4) Theory and numerical simulations: Analytical theory, numerical simulation, comparison of theoretical with experimental results, and modification of theoretical approaches, models, grids, etc., have been conducted for several complicated 2D and 3D nozzle and inlet designs using NASA, ICASE, and IM/MSU codes based on full Euler and Navier-Stokes solvers: CFL3D, FLUENT, and GODUNOV, and others; (5) Experimental Tests: (a) A new course: "Advanced Aerodynamics and Aircraft Performance" presented in spring semester, 2001; training and experimental test research using the HU LSWT. (b) Small-scale M6bius-shaped screws were tested in different conditions and their application has shown essential benefits by comparison with traditional designs; (6) Installation in the FM&AL computer system: second software TECPLOT 8.0 for the UNIX SGI workstation and free TECPLOT 7.5 for the PC Dell computer, and 2D and 3D GRIDGEN (version 9) for the UNIX SGI as well as installation of two free NASA codes, 3D MAG and VULCAN; (7) Student Research Activity: Involvement of two undergraduate students as research assistants in the current research project.

Gilinsky, Mikhail↗

A comparison of theoretical and experimental pressure distributions for two advanced fighter wings

A comparison was made between experimental pressure distributions measured during testing of the Vought A-7 fighter and the theoretical predictions of four transonic potential flow codes. Isolated wind and three wing-body codes were used for comparison. All comparisons are for transonic Mach numbers and include both attached and separate flows. In general, the wing-body codes gave better agreement with the experiment than did the isolated wing code but, because of the greater complexity of the geometry, were found to be considerably more expensive and less reliable.

Haney, H. P.↗

Investigation of Advanced Counterrotation Blade Configuration Concepts for High Speed Turboprop Systems. Task 8: Cooling Flow/heat Transfer Analysis User's Manual

The focus of this task was to validate the ADPAC code for heat transfer calculations. To accomplish this goal, the ADPAC code was modified to allow for a Cartesian coordinate system capability and to add boundary conditions to handle spanwise periodicity and transpiration boundaries. This user's manual describes how to use the ADPAC code as developed in Task 5, NAS3-25270, including the modifications made to date in Tasks 7 and 8, NAS3-25270.

Hall, Edward J.↗

Peregrine Software Development: Report on the Code Conversion From Python to C++

This work package seeks to convert the Peregrine software tool from its original Python implementation to a production version based on the C++ language. Peregrine is a powerful research platform with a multitude of advanced data analytics and data visualization functionalities. Developed by scientists to explore multimodal and multidimensional data related to the production of components using powder bed additive manufacturing processes, the tool implements state-of-the-art algorithms to assist machine users in making build or part quality determinations. Given that Peregrine is data-intensive, the goal of this conversion is to enhance the tool’s flexibility and interactivity and reduce the number of code dependencies to facilitate its deployment as part of the ongoing technology transfer campaign. This brief document provides an overview of Peregrine’s functionalities and capabilities, along with a detailed description of the core functionalities that have been implemented to date in the new C++ version. This document serves as a development update at the end of the first year of the ongoing conversion and will be regularly updated as progress continues.

97 MATHEMATICS AND COMPUTING↗

Electric Vehicle Charging for Residential and Commercial Energy Codes: Technical Brief

Numerous studies show that sales of electric vehicles (EVs) have grown consistently over recent years in the U.S. The U.S. Energy Information Administration (EIA) estimated 3 million EVs were on the road in 2022, and the Edison Electric Institute (EEI) forecasts a total of 26.4 million EVs on the road by 2030. Based on this forecast, EEI projects the need for an additional 12.9 million EV charge ports by 2030. If EV charging infrastructure fails to keep pace with sales of EVs it could result in consumers stranded without options to power their vehicles. EVs are capable of providing substantial benefits to the consumers. EVs are less expensive to operate than conventional internal combustion engine vehicles, have lower maintenance costs, and have the convenience of fueling (charging) at home or work. Studies conducted in California show that costs associated with installing EV charging infrastructure can be substantially more expensive for retrofit scenarios compared to new construction, making inclusion of EV infrastructure in new construction codes a cost-effective policy option to increase infrastructure to meet growing demands. PNNL tracks adoption of mandatory EV provisions across the U.S. As of December 20, 2024, 12 states (California, Oregon, Washington, Colorado, New Mexico, Illinois, Maryland, Delaware, New Jersey, Rhode Island, Massachusetts and Vermont) and 53 local governments have added EV provisions to their building codes, local ordinances and zoning requirements. Originally published in 2022, this tech brief has been revised to align with recent model energy code committee discussions and published EV infrastructure code language. This technical brief summarizes market trends, costs and benefits, and provides sample code language for EV charging infrastructure for consideration to be included in model codes, such as the International Energy Conservation Code (IECC) and ANSI/ASHRAE/IES Standard 90.1, as well as directly by states and local governments in their building codes. The technical brief summarizes related efforts undertaken by states and local governments, and builds upon language considered during the 2021 and 2024 IECC development cycles.

2021 IECC↗

As-Run Physics Analysis for the AGC-4 Experiment Irradiated in the ATR

This Engineering Calculations Analysis Report (ECAR) documents the results of the Advanced Test Reactor (ATR) detailed physics analyses performed to calculate the displacements per atom (DPA) and the fast neutron fluence (E > 0.1 MeV) of the Advanced Graphite Creep (AGC) experiment, AGC-4, irradiated in the ATR East Flux Trap (EFT) (see Figure 1) during ATR Cycle 157D, 158A, 162A, 162B, 164A, 164B, 166A, and Cycle 166B. This ECAR also reports the neutron and photon heat rates for the materials of the AGC-4 experiment for ATR Cycle 158A (timestep 19), which provides to the maximum heating. The results for these evaluations and analysis are reported herein. The AGC-4 as-run specimen neutron fast fluence (E > 0.1 MeV), DPA, and material heat rate calculations were performed using a general-purpose Monte Carlo N-Particle (MCNP) code. All calculated results are tabulated herein.

11 NUCLEAR FUEL CYCLE AND FUEL MATERIALS↗

Code interoperability extends the scope of quantum simulations

The functionality of many materials is critically dependent on the integration of dissimilar components and on the interfaces that arise between them. The description of such heterogeneous components requires the development and deployment of first principles methods, coupled to appropriate dynamical descriptions of matter and advanced sampling techniques, in order to capture all the relevant length and time scales of importance to the materials’ performance. It is thus essential to build simple, streamlined computational schemes for the prediction and design of multiple properties of broad classes of materials, by developing interoperable codes which can be efficiently coupled to each other to perform complex tasks. We discuss the use of interoperable codes to simulate the structural and spectroscopic characterization of materials, including chemical reactions for catalysis, the description of defects for quantum information science, and heat and charge transport.

36 MATERIALS SCIENCE↗

Celeritas: Accelerating Geant4 with GPUs

Celeritas [1] is a new Monte Carlo (MC) detector simulation code designed for computationally intensive applications (specifically, High Lumi- nosity Large Hadron Collider (HL-LHC) simulation) on high-performance heterogeneous architectures. In the past two years Celeritas has advanced from prototyping a GPU-based single physics model in infinite medium to implementing a full set of electromagnetic (EM) physics processes in complex geometries. The current release of Celeritas, version 0.3, has incorporated full device-based navigation, an event loop in the presence of magnetic fields, and detector hit scoring. New functionality incorporates a scheduler to offload electromagnetic physics to the GPU within a Geant4-driven simulation, enabling integration of Celeritas into high energy physics (HEP) experimental frameworks such as CMSSW. On the Summit supercomputer, Celeritas performs EM physics between 6 and 32 faster using the machine’s Nvidia GPUs compared to using only CPUs. When running a multithreaded Geant4 ATLAS test beam application with full hadronic physics, using Celeritas to accelerate the EM physics results in an overall simulation speedup of 1.8–2.3× on GPU and 1.2× on CPU.

Johnson, Seth R.↗

Challenges in the designing, planning and deployment of hydrogen refueling infrastructure for fuel cell electric vehicles

Hydrogen can power transportation with near zero greenhouse gas emissions. With government support, early market development is now underway in several nations thanks to technological advances in fuel cell vehicles and electricity generation from renewable energy. Deploying a sustainable hydrogen refueling infrastructure faces methodological and practical challenges ranging from the creation of appropriate codes to managing the co-evolution of the refueling network and growth of the stock of hydrogen fuel cell vehicles. Furthermore, this paper presents a comprehensive review of the challenges facing the designing, planning and deployment of hydrogen refueling infrastructure progress to date and outlook for the future. The design and costs of refueling infrastructure as well as the lifecycle environmental effects of hydrogen vehicles depend on how hydrogen is produced and delivered to refueling stations. In recent years, important advances have been made in methods for planning the numbers, sizes and location of hydrogen stations. Institutional barriers are also gradually being overcome. Co-evolving the deployment of stations and the demand for fuel cell vehicles remains a crucial subject for future research.

33 ADVANCED PROPULSION SYSTEMS↗

Dynamic and Responsive Distributed Energy Resource Education Solutions for Building, Fire, and Safety Department Officials (Final Technical Report)

From April 2021 through March 2024, the Interstate Renewable Energy Council (IREC) led a collaborative project to develop a free online clearinghouse of educational resources about solar photovoltaics (PV), energy storage systems (ESS), electric vehicle supply equipment (EVSE), and grid-interactive efficient building (GEB) technologies. Two websites—the Clean Energy Clearinghouse and CleanEnergyTraining.org—housed over 70 educational resources. Over the course of the three-year project, 154,272 unique visitors accessed the learning materials. Learner feedback was overwhelmingly positive. Even through the end of the project, there was sustained demand for education and communication. A primary innovation of the project was to drive multiple complementary audiences to the same place. Building owners, designers, installation contractors and developers, authorities having jurisdiction (AHJs), and fire service personnel all benefit from a shared understanding of clean energy technologies, including safety and code-related requirements. When considering the impact on the target audience, the project team worked with partners and advisors to inform resource creation and delivery in such a way as to address key motivational factors of the target audience and compel each user to seek additional information on the topic and return to the Clean Energy Clearinghouse website as their central location for more information. Resources were intentionally developed to be concise—five to 15 minutes—and accessible, meaning not overly technical. Providing basic information demystified the technologies and invited the professional to explore additional learning opportunities. Awardee and partner collaboration was key to project success. IREC facilitated collaboration among the other Topic 2 awardees, Southface and New Buildings Institute (NBI). The three awardees shared relevant information gained through discovery and validation questionnaires that informed product development and reduced duplication of effort by coordinating the development of complementary, and not competing, educational resources. Inspired by this collaboration, IREC brought on additional partners even in the final year of the project. Five regional energy efficiency organizations were part of the project, which expanded the connection between efficiency and distributed energy resources. We also included resources on the Clearinghouse that were developed through other federally funded projects, such as the Buildings Energy Efficiency Frontiers & Innovation Technologies (BENEFIT) program. The website was developed with the learner in mind, and not solely the funding source. Feedback from stakeholders throughout the project, and especially in its final year, indicated the need for continued education and facilitated communication among stakeholders to further the safe and widespread adoption of clean energy.

14 SOLAR ENERGY↗

Advanced Reactor Safeguards: 2022 Program Roadmap

The Advanced Reactor Safeguards (ARS) program was established in 2020 as part of appropriations for the Advanced Reactor Demonstration Program (ARDP) through the Office of Nuclear Energy in the Department of Energy. The goal of this program is to help address near term challenges that advanced nuclear reactor vendors face in meeting domestic Material Control and Accountancy (MC&A) and Physical Protection System (PPS) requirements for U.S. construction. The technical work in the program is meant to (1) support nuclear reactor vendors with advanced MC&A and PPS designs for next generation reactors, (2) provide technical bases for the regulator, and (3) promote the integration of Safeguards and Security by Design early in the design process. Existing domestic regulations for safeguards and security, as outlined in the Code of Federal Regulations, were written for large light water reactors, and rule-making efforts are underway to develop regulations more suited to different reactor designs. The ARS program seeks to remove roadblocks in the deployment of new and advanced reactors by solving regulatory challenges, reducing safeguards and security costs, and utilizing the latest technologies and approaches for robust plant monitoring and protection. This roadmap discusses the goals of the ARS program, current research, and program plan for the next five years.

98 NUCLEAR DISARMAMENT, SAFEGUARDS, AND PHYSICAL P↗

UQpy: A general purpose Python package and development environment for uncertainty quantification

In this paper, we present the UQpy software toolbox, an open-source Python package for general uncertainty quantification (UQ) in mathematical and physical systems. The software serves as both a user-ready toolbox that includes many of the latest methods for UQ in computational modeling and a convenient development environment for Python programmers advancing the field of UQ. The paper presents an introduction to the software's architecture and existing capabilities, divided in the code in a set of modules centered around different UQ tasks such as sampling methods, generation of random processes and random fields, probabilistic inverse modeling, reliability analysis, surrogate modeling, and active learning. The paper also highlights the importance of the RunModel module, which is used to drive simulations in the uncertainty analyses performed in UQpy. This module conveniently allows the user to define computational models directly in Python, or to run simulations from a third-party software in serial or in parallel. To illustrate the various capabilities, two examples are tracked throughout the paper and analyzed repeatedly for various UQ tasks. The first is a Python model solving a nonlinear structural dynamics problem, used to illustrate UQpy's capabilities in sampling and forward propagation of high dimensional random vectors (stochastic processes), and probabilistic inference. The second model is a third-party Abaqus finite element model solving the thermomechanical response of a beam structure. This example is used to illustrate UQpy's capabilities in variance reduction sampling techniques, reliability analysis, surrogate modeling and active learning techniques.

97 MATHEMATICS AND COMPUTING↗

Profiling expression strategies for a type III polyketide synthase in a lysate-based, cell-free system

Abstract Some of the most metabolically diverse species of bacteria (e.g., Actinobacteria) have higher GC content in their DNA, differ substantially in codon usage, and have distinct protein folding environments compared to tractable expression hosts like Escherichia coli . Consequentially, expressing biosynthetic gene clusters (BGCs) from these bacteria in E. coli often results in a myriad of unpredictable issues with regard to protein expression and folding, delaying the biochemical characterization of new natural products. Current strategies to achieve soluble, active expression of these enzymes in tractable hosts can be a lengthy trial-and-error process. Cell-free expression (CFE) has emerged as a valuable expression platform as a testbed for rapid prototyping expression parameters. Here, we use a type III polyketide synthase from Streptomyces griseus , RppA, which catalyzes the formation of the red pigment flaviolin, as a reporter to investigate BGC refactoring techniques. We applied a library of constructs with different combinations of promoters and rppA coding sequences to investigate the synergies between promoter and codon usage. Subsequently, we assess the utility of cell-free systems for prototyping these refactoring tactics prior to their implementation in cells. Overall, codon harmonization improves natural product synthesis more than traditional codon optimization across cell-free and cellular environments. More importantly, the choice of coding sequences and promoters impact protein expression synergistically, which should be considered for future efforts to use CFE for high-yield protein expression. The promoter strategy when applied to RppA was not completely correlated with that observed with GFP, indicating that different promoter strategies should be applied for different proteins. In vivo experiments suggest that there is correlation, but not complete alignment between expressing in cell free and in vivo. Refactoring promoters and/or coding sequences via CFE can be a valuable strategy to rapidly screen for catalytically functional production of enzymes from BCGs, which advances CFE as a tool for natural product research.

59 BASIC BIOLOGICAL SCIENCES↗